ABSTRACT Lung cancer is the leading cause of cancer-related death in the world with a 5-year survival rate of less than 15% which has not changed for the past three decades. At diagnosis, most patients already have advanced-stage disease and the use of chemotherapy and radiotherapy is rarely curative. This poor overall survival may be due to the fact that both chemotherapy and radiation therapy preferentially kill actively dividing cancer cells, which survive such treatment and eventually cause cancer recurrence and death. Thus, novel approaches to target quiescent tumor cells are needed. The cancer stem cell hypothesis proposes that cancers arise from relatively inactive stem cells that are capable of self-renewal, proliferation, and differentiation into more mature cancer cells. Evidence supports the existence of cancer stem cells in several tumor types and recent studies have identified putative stem cells in lung cancer. We have been evaluating the biologic characteristics of lung cancer stem cells in an effort to develop novel strategies for treatment that would reduce recurrence rates and improve patient survival. Notch is a highly conserved signaling pathway that regulates cell fate decisions during development and in tissue homeostasis. Notch signaling regulates the self-renewal potential of stem/progenitor cell populations in multiple contexts and has been proposed to support the self-renewal capability of some cancer stem cell populations. Gene expression profiling of non-small-cell lung cancer specimens has shown elevated expression of downstream effectors of Notch signaling, suggesting an important role of Notch signaling in lung carcinogenesis. Data from our laboratory and from other investigators have shown that inhibition of Notch signaling by gamma secretase inhibitors (GSI) can decrease cell proliferation and induce apoptosis in some lung cancer cell lines. However, the specific mechanisms underlying this activity of GSI and the effect of Notch inhibition on lung cancer and lung cancer stem cells have not been explored. Our preliminary data show that different lung cancer cell lines have variable expression of Notch receptors and its pathway components. In these cell lines, the GSI MRK003 induces growth inhibition due to cell cycle arrest leading to apoptotic cell death. In addition, treating lung cancer mouse xenografts with MRK003 limits the serial reimplantation potential of these tumors, suggesting a possible inhibitory effect on cancer stem cells. Thus, it is important to distinguish global effects of the drug on cancer cells, and the specific effects on cancer stem cells. In this application, we propose to investigate the specific role of the Notch pathway in lung cancer stem cells utilizing primary human lung tumor tissue and assess the impact of Notch inhibition on tumor formation. This work presents a novel approach in treating lung cancer by targeting cancer stem cells and provides basis for future clinical trials.